The long-term objective of this project is to develop material systems that promote new bone formation for reconstructive dental and craniofacial applications. The overall hypothesis guiding our research is that material systems may be designed, based on a fundamental and quantitative understanding of cell receptormaterial ligand binding, to provide a selective advantage to specific cell types present in a mixture, and guide tissue formation from these cells. This hypothesis represents a continuation of our current effort to develop materials to promote bone regeneration by understanding and manipulating the role of cell adhesion peptide presentation (type, density, spacing of ligand, and mechanical properties of presenting substrate) in osteoprogenitor differentiation and bone formation. The specific hypothesis to be tested in the extension of this MERIT award is that the mechanical properties and cell adhesion ligand presentation of alginate gels may be tuned to specifically promote mesenchymal stem cell (MSC) adhesion, proliferation and differentiation, and enhance bone regeneration in vivo from transplanted cell populations or induced host cells. The specific aims will include: (1) Develop a FRET-based technique to perform single cell analysis of receptor-ligand binding dynamics (kon, koff, KD) in order to determine how ligand presentation (density, spacing, gel mechanical properties) regulate bond formation, (2) Analyze kon, koff, and KD with this technique using libraries of synthetic peptides derived from natural extracellular matrix molecules (e.g., fibronectin, collagen, laminin) in order to identify materials with high specificity for MSC adhesion and possibly distinct stages of differentiation, and (3) Test the ability of specific ligands and ligand presentations chosen from the large-scale screening to enhance bone regeneration from transplanted cell populations and host cell populations. Success in these studies will lead to biomaterials that can dramatically enhance bone regeneration by specifically promoting the adhesion, proliferation and differentiation of the small numbers of osteoprogenitors typically present in cell populations, either in vitro or in vivo.